PROJECT SUMMARY/ABSTRACT Loading peptides onto major histocompatibility complex (MHC) class II molecules involves the actions HLA-DM (DM) and HLA-DO (DO). DM regulates the exchange of MHC-II-associated invariant chain peptide with other peptides, while DO acts to inhibit interaction of DM with MHC-II. New high-resolution microscopy results from our lab revealed that DM localization is altered at the presence of DO, and additional data demonstrated a change in specific MHC-II peptide presentation at different DO/DM ratios. The major hypotheses addressed herein are that: (1) DO dominantly directs the DM subcellular destination to the low pH compartment where DO is being irreversibly degraded. (2) Changes in the DO/DM ratio can affect both quantity and quality of the peptides that are being displayed via MHC-II. The specific aims to be studied are: Aim 1: Delineate the subcellular localization of the DO/DM complexes as controlled by DO The subcellular localization of DM and DO will be evaluated in a genetically engineered cell line system, with cell lines that express DM with different levels of DO or altered DO localization motifs. (1) high-resolution microscopy and (2) subcellular fractionation followed by western blot analysis will be applied to determine the subsequent DO/DM localization. Aim 2: Characterize the peptide repertoire that is being displayed on surface MHC-II at different DO/DM levels The peptide repertoire of cell lines genetically engineered to express different levels of DM and/or DO will be evaluated by mass spectrometry. The output of the peptides extracted under different DO/DM ratios will be analyzed to assess their structural/biophysical properties and their protein (intracellular compartment) source. Additionally, the affinity of these peptides to the MHC-II allele expressed in this cell line will be measured as an IC50 value in competitive peptide binding assays. The overall objective of this study is to gain a more precise understanding of how the changes in the DO/DM ratio regulate the antigen presentation process in MHC class II+ cells. Understanding of this mechanism may suggest strategies to modulate acquired tolerance by regulatory T cells and antigen presentation to T effector cells.